# Patent

## Title
Expression system

## Abstract

The present invention relates generally to methods and compositions for expression of polypeptides or delivery of interfering RNAs in various cell types.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 11/628,374, filed Oct. 17, 2007, which is a National Stage application under 35 U.S.C. §371 of International Application. No. PCT/US2005/019371, filed Jun. 1, 2005, which claims benefit of priority from U.S. Provisional Application Ser. No. 60/576,169, filed Jun. 1, 2004. The disclosures of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.


TECHNICAL FIELD
The present invention relates generally to methods and compositions for a novel expression system. The system may be used for the expression of polypeptides in various cell types or to deliver interfering RNAs.
BACKGROUND
DNA cloning technology provides a readily amplifiable source of genes encoding any protein of interest. When the recombinant protein itself is needed, genes are cloned in expression vectors that are introduced into appropriate cell types where protein synthesis can take place. To produce large amounts of these proteins, two general types of transient gene expression vectors have been used: plasmid DNA vectors which are introduced directly into cells and viral vectors that express foreign genes as part of their genetic material. The latter type of vector is generally more efficient in higher eukaryotic cells because all cells can be infected simultaneously and many viruses can express proteins at very high levels. Plasmid vector preparation is less labor intensive but DNA transfection can be inherently less efficient and amounts of protein synthesized are generally lower.
High-level recombinant protein expression is crucial for the biopharmaceutical industry as well as for basic research. Large amounts of specific proteins are very often required for general biochemical characterization, structural studies, drug discovery development, gene therapy, subunit vaccine production, and reagent use. Different uses dictate which particular protein expression system provides the best combination of properties. For example, high-level transient protein expression in mammalian cells most often makes use of viral vectors (e.g., adenovirus, baculovirus, poxvirus, alphavirus). In most applications, the gene of interest is cloned into the virus genome or a derivative replicon which is labor intensive and time consuming. Plasmid vectors are also used for transient protein expression but efficiency is generally much lower. Another method employs recombinant viruses that express the T7 RNA polymerase to drive expression of desired proteins from plasmids under control of a T7 promoter. This latter approach is very efficient using a vaccinia-T7 recombinant virus especially when incorporating an internal ribosome entry sequence (IRES) in the T7 transcript. However, high level protein production using the vaccinia-T7 system is limited to host cells that grow the virus efficiently. Moreover, the use of an infectious virus related to the smallpox vaccine strain raises biosafety concerns.
In the last few years, advances in nucleic acid chemistry and gene transfer have inspired new approaches to engineer specific interference with gene expression. Antisense technology has been the most commonly described approach in protocols to achieve gene-specific interference. For antisense strategies, stoichiometric amounts of single-stranded nucleic acid complementary to the messenger RNA for the gene of interest are introduced into the cell. Some difficulties with antisense-based approaches relate to delivery, stability, and dose requirements. In general, cells do not have an uptake mechanism for single-stranded nucleic acids, hence uptake of unmodified single-stranded material is extremely inefficient. While waiting for uptake into cells, the single-stranded material is subject to degradation. Because antisense interference requires that the interfering material accumulate at a relatively high concentration (at or above the concentration of endogenous mRNA), the amount required to be delivered is a major constraint on efficacy. As a consequence, much of the effort in developing antisense technology has been focused on the production of modified nucleic acids that are both stable to nuclease digestion and able to diffuse readily into cells.
Double-stranded RNA (dsRNA) can induce sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). However, in mammalian cells, dsRNA that is 30 base pairs or longer can induce sequence-nonspecific responses that trigger a shut-down of protein synthesis. Recent work suggests that RNA fragments are the sequence-specific mediators of RNAi (Elbashir et al., Nature 2001 411:494; Elbashir et al., Genes and Development 2001, 15:188). Interference of gene expression by these small interfering RNA (siRNA) is now recognized as a naturally occurring strategy for silencing genes in C. elegans, Drosophila, plants, and in mouse embryonic stem cells, oocytes and early embryos (Cogoni et al., Antonie Van Leeuwenhoek 1994, 65:205; Baulcombe, Plant Mol. Biol., 1996, 32:79; Kennerdell and Carthew, Cell 1998, 95:1017; Timmons and Fire, Nature 1998, 395:854; Waterhouse et al., Proc. Natl. Acad. Sci. U.S.A. 1998, 95:13959; Wianny and Zernicka-Goetz, Nat. Cell Biol. 2000, 2:70; Yang et al., Mol. Cell Biol. 2001, 21:7807; Svoboda et al., Development 2000, 127:4147 (2000). In mammalian cell culture, a siRNA-mediated reduction in gene expression has been accomplished by transfecting cells with synthetic RNA oligonucleotides (Caplan et al., Proc. Natl. Acad. Sci. U.S.A. 2001, 98:9742; Elbashir et al., 2001, supra). However, as Bass (Nature 2001, 411:428) notes, various issues regarding the use of siRNA in mammalian cells have yet to be addressed, including effective delivery of siRNA to mammalian cells in vivo. Furthermore, if siRNA is to be utilized in in vivo therapy, it will be important in many cases to develop methods to express siRNA in tissues in vivo to achieve extended intracellular transcription of the siRNA.
Vesicular stomatitis virus (VSV), of the genus, Vesiculovirus, is the prototypic member of the family Rhabdoviridae, and is an enveloped virus with a negative stranded RNA genome that causes a self-limiting disease in live-stock and is essentially non-pathogenic in humans. Balachandran and Barber (2000, IUBMB Life 50: 135-8). Rhabdoviruses have single, negative-strand RNA genomes of 11,000 to 12,000 nucleotides (Rose and Schubert, 1987, Rhabdovirus genomes and their products, in The Viruses: The Rhabdoviruses, Plenum Publishing Corp., NY, pp. 129-166). The virus particles contain a helical, nucleocapsid core composed of the genomic RNA and protein. Generally, three proteins, termed N (nucleocapsid, which encases the genome tightly), P (formerly termed NS, originally indicating nonstructural), and L (large) are found to be associated with the nucleocapsid. An additional matrix (M) protein lies within the membrane envelope, perhaps interacting both with the membrane and the nucleocapsid core. A single glycoprotein (G) species spans the membrane and forms the spikes on the surface of the virus particle. Glycoprotein G is responsible for binding to cells and membrane fusion. The VSV genome is the negative sense (i.e., complementary to the RNA sequence (positive sense) that functions as mRNA to directly produce encoded protein), and rhabdoviruses must encode and package an RNA-dependent RNA polymerase in the virion (Baltimore et al., 1970, Proc. Natl. Acad. Sci. USA 66: 572-576), composed of the P and L proteins. This enzyme transcribes genomic RNA to make subgenomic mRNAs encoding the 5-6 viral proteins and also replicates full-length positive and negative sense RNAs. The genes are transcribed sequentially, starting at the 3′ end of the genomes.
VSV replicates rapidly (<12 hours) and very efficiently in the cytoplasm of almost all vertebrate cells and produces very high levels of infectious virus (titers approaching 20,000 infectious units/cell in some cases) and can also infect insect cells. The sequences of the VSV mRNAs and genome are described in Gallione et al. 1981, R Virol. 39: 529-535; Rose and Gallione, 1981, J. Virol. 39: 519-528; Rose and Schubert, 1987; Rhabdovirus genomes and their products, p. 129-166, in R. R. Wagner (ed.); The Rhabdoviruses, Plenum Publishing Corp., NY; Schubert et al., 1985, Proc. Natl. Acad. Sci. USA 82: 7984-7988). WO96/34625 published Nov. 7, 1996, disclose methods for the production and recovery of replicable vesiculovirus. U.S. Pat. No. 6,168,943, issued Jan. 2, 2001, describe methods for making recombinant vesiculoviruses.
Growth in tissue culture and purification of virus is relatively simple and methods for engineering mutations or additional genes in the virus genome, while retaining very high infectivity, are well established. It is moreover possible to engineer the surface protein of the virus and target infection to specific cell types. Natural hosts include cattle, horses, and pigs where it causes a non-fatal but debilitating disease. Laboratory strains pose very little if any risk of pathogenicity in humans. VSV is currently being explored as a vector for vaccine production, gene replacement therapy, and anticancer therapy.
Expression of the T7 RNA polymerase enzyme by recombinant viruses has been reported (see e.g. Mohammed et al., Methods Mol Biol. 2004; 269:41-50; and Eckert et al., J Gen Virol. 1999 June; 80 (Pt 6):1463-9). Recombinant T7-expressing viruses are capable of driving transient expression of proteins from plasmids. The best characterized of these systems is the recombinant vaccinia-T7 virus which yields very high levels or protein. The limitations and uses of any of the virus-T7 expression systems are in large part governed by the properties of the virus. Thus, there is a need for a safe and efficient alternative virus-T7 polypeptide expression system.
SUMMARY
Expression systems comprising a combination of a virus vector and a plasmid that leads to very high transient expression of polynucleotides and polypeptides are provided. The system may also be used to deliver interfering RNAs. Methods for making and using the system are also provided. Vesicular stomatitis virus was engineered to express the prokaryotic T7 RNA polymerase enzyme in the cytoplasm of infected cells. Infected cells were then transfected with a plasmid DNA encoding a gene of interest downstream of a T7 promoter sequence and an internal ribosome entry sequence. This results in cytoplasmic accumulation of large amounts of T7 mRNA transcripts which are efficiently translated into the desired protein. Methods and compositions are also provided bypassing the need for an internal ribosome entry sequence in the transfected plasmid. Provided herein are compositions and methods for a recombinant vesicular stomatitis virus vector expression system. A vesicular stomatitis virus vector particle (VSV) encoding a T7 RNA polymerase is provided and used to infect a cell, such that the T7 polymerase is expressed in the cell. Also provided is a recombinant plasmid vector encoding a heterologous gene under control of a T7 promoter and also encoding an IRES element. Cells infected with VSV-T7 viral particles are subsequently transfected with the recombinant plasmid vector such that a polynucleotide is expressed from the transcripts encoded by the heterologous gene in the cell. A method for producing a polypeptide by contacting cells with the recombinant vesicular stomatitis virus vector expression system is also provided. In one embodiment, the VSV-T7 viral particles include the sequences of polynucleotides as set forth in SEQ ID NO:1. In another embodiment, the M and G genes of the VSV-T7 vector virus vector particle are deleted or mutated such that the virus vector particle is replication-deficient. A recombinant plasmid vector consisting essentially of the polynucleotide sequence as set forth in SEQ ID NO:3 are also provided.
In one aspect, the invention provides a recombinant vesicular stomatitis virus vector expression system comprising:
(a) a vesicular stomatitis virus vector particle (VSV) comprising a polynucleotide encoding a T7 RNA polymerase, the vector particle being used to infect a cell, such that the T7 polymerase is expressed in the cell; and
(b) a recombinant plasmid vector comprising a polynucleotide comprising the following elements operably linked 5′ to 3′:

    
    
        (i) a bacteriophage promoter, and
        (ii) a heterologous gene, wherein the heterologous gene comprises a sequence encoding an internal ribosome entry site (IRES), the recombinant plasmid vector being used to transfect said cell so that a polypeptide is expressed from transcripts encoded by said heterologous gene in the cell. The promoter may be a T7 promoter. A method for producing a polypeptide comprising contacting cells with this recombinant vesicular stomatitis virus vector expression system is also provided.
    
    


The vesicular stomatitis virus vector particle may comprise the polynucleotide sequence in SEQ ID NO:1, and the M and G genes of the virus vector particle may be deleted or mutated such that the virus vector particle is replication-deficient. The recombinant plasmid vector may comprise a T7 promoter as set forth in SEQ ID NO:3 corresponding to nucleotide positions 794 to 813, and a heterologous gene.
In another aspect, the invention provides a recombinant vesicular stomatitis virus vector expression system comprising:
(a) a vesicular stomatitis virus vector particle (VSV) comprising a polynucleotide encoding a T7 RNA polymerase, the vector particle being used to infect a cell, such that the T7 polymerase is expressed in the cell;
(b) a first recombinant plasmid vector comprising a polynucleotide comprising the following elements operably linked 5′ to 3′:

    
    
        (i) a bacteriophage promoter, and
        (ii) a heterologous gene, the recombinant plasmid vector being used to transfect said cell so that a polypeptide is expressed from transcripts encoded by said heterologous gene in the cell;
    
    


(c) a second recombinant plasmid vector comprising the D1 catalytic subunit of vaccinia capping enzyme (D1) as set forth in SEQ ID NO:5; and
(d) a third recombinant plasmid vector comprising the D12 subunit of vaccinia capping enzyme (D12) as set forth in SEQ ID NO:6. The promoter may be a T7 promoter. A method for producing a polypeptide comprising contacting cells with this recombinant vesicular stomatitis virus vector expression system is also provided.
The first recombinant plasmid vector may comprise a T7 promoter as set forth in SEQ ID NO:3 corresponding to nucleotide positions 794 to 813, and a heterologous gene, and wherein the IRES polynucleotide encoding sequences are deleted. The vesicular stomatitis virus vector particle may comprise the polynucleotide sequence in SEQ ID NO:1 and the M and G genes of the virus vector particle are deleted or mutated such that the virus vector particle is replication-deficient. The second and third recombinant plasmid vectors may encode the sequences for D1 and D12 catalytic subunits of vaccinia virus-capping enzyme as set forth in SEQ ID NOS:5 and 6.
In yet another aspect, the invention provides a method of producing a vesicular stomatitis virus vector particle, said method comprising:
(a) transfecting a permissive producer cell with a vector comprising a nucleic sequence of at least part of the VSV genome and T7 RNA polymerase; and
(b) growing said producer cell under cell culture conditions sufficient to allow producing of vesicular stomatitis virus vector particles in said cell; and
(c) collecting said particles from said producer cell. The producer cell may be grown in cell culture medium, and the vector particles may be collected from the medium.
In yet a further aspect, a method for producing replication-defective vesicular stomatitis virus vector particles said method comprising:
(a) transfecting a permissive producer cell with a vector comprising a nucleic acid sequence of at least part of the VSV genome and T7 RNA polymerase wherein the M and G genes are deleted; and
(b) growing said producer cell under cell culture conditions sufficient to allow producing of vesicular stomatitis virus vector particles in said cell; and
(c) co-transfecting said cell with plasmids encoding M and G genes; and
(d) collecting said particles from said producer cell. The producer cell may be grown in cell culture medium, and the replication-defective vector particles may be collected from said medium.
In a still further aspect, the invention provides recombinant vesicular stomatitis virus vector expression system comprising:
(a) a vesicular stomatitis virus vector particle (VSV) comprising a polynucleotide encoding a T7 RNA polymerase, the vector particle being used to infect a cell, such that the T7 polymerase is expressed in the cell; and
(b) a recombinant plasmid vector comprising a polynucleotide comprising the following elements operably linked 5′ to 3′:

    
    
        (i) a bacteriophage promoter, and
        (ii) a heterologous gene, wherein the heterologous gene comprises a sequence encoding an internal ribosome entry site (IRES), the recombinant plasmid vector being used to transfect said cell so that an interfering RNA polynucleotide is expressed from said heterologous gene in the cell. The promoter may be a T7 promoter. A method for delivering an interfering RNA polynucleotide into target cells comprising contacting cells with this recombinant vesicular stomatitis virus vector expression system is also provided.
    
    


The vesicular stomatitis virus vector particle may comprise the polynucleotide sequence in SEQ ID NO:1, and the M and G genes of the virus vector particle are deleted or mutated such that the virus vector particle is replication-deficient. The recombinant plasmid vector may comprise a T7 promoter as set forth in SEQ ID NO:3 corresponding to nucleotide positions 794 to 813, and a heterologous gene.
Another aspect of the invention provides a recombinant vesicular stomatitis virus vector expression system comprising:
(a) a vesicular stomatitis virus vector particle (VSV) comprising a polynucleotide encoding a T7 RNA polymerase, the vector particle being used to infect a cell, such that the T7 polymerase is expressed in the cell;
(b) a first recombinant plasmid vector comprising a polynucleotide comprising the following elements operably linked 5′ to 3′:

    
    
        (i) a bacteriophage promoter, and
        (ii) a heterologous gene, the recombinant plasmid vector being used to transfect said cell so that an interfering RNA polynucleotide is expressed from said heterologous gene in the cell;
    
    


(c) a second recombinant plasmid vector comprising the D1 catalytic subunit of vaccinia capping enzyme (D1) as set forth in SEQ ID NO:5; and
(d) a third recombinant plasmid vector comprising the D12 subunit of vaccinia capping enzyme (D12) as set forth in SEQ ID NO:6. The promoter may be a T7 promoter. A method for delivering an interfering RNA polynucleotide into target cells comprising contacting cells with this recombinant vesicular stomatitis virus vector expression system is also provided.
The first recombinant plasmid vector may comprise a T7 promoter as set forth in SEQ ID NO:3 corresponding to nucleotide positions 794 to 813, and a heterologous gene, and wherein the IRES polynucleotide encoding sequences are deleted. The vesicular stomatitis virus vector particle may comprise the polynucleotide sequence in SEQ ID NO:1, and the M and G genes of the virus vector particle are deleted or mutated such that the virus vector particle is replication-deficient. The second and third recombinant plasmid vectors may encode the sequences for D1 and D12 catalytic subunits of vaccinia virus-capping enzyme as set forth in SEQ ID NOS:5 and 6.
The invention also provides any of the above methods for expressing proteins or delivering interfereing RNA wherein the cells are transfected with said recombinant plasmid vectors by a suitable method such as liposome mediated transfer, lipofection, polycation-mediated transfer, or direct DNA transfer or uptake. The expression of a gene product in a cell may be reduced as a result of expression of an interfering RNA polynucleotide.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.



DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts a pVSV-T7 plasmid map.
FIG. 2 depicts a pVSV-GFP plasmid map.
FIG. 3 depicts a pTM1-GFP plasmid map.
FIG. 4 depicts a pSP73-GFP plasmid map.
FIG. 5 depicts a pTM1-D1 plasmid map.
FIG. 6 depicts a pTM1-D12 plasmid map.
FIG. 7 represents a schematic of a vesicular stomatitis virus; structural genes are shown as is a schematic of the VSV genome.
FIG. 8 represents a schematic of the VSV genome, including the sequences of the leader-N gene junction (UUGAAAUUGUCNNAG; SEQ ID NO:7) and other gene junctions (AUACUUUUUUUG/CAUUGUCNNAG; SEQ ID NO:8). Unique features of VSV transcription are also provided.
FIG. 9 represents a schematic of general features of VSV RNA synthesis, including a schematic of the VSV genome showing the sequences of the leader-N gene junction (SEQ ID NO:7) and other gene junctions (SEQ ID NO:8).
FIG. 10 represents a schematic VSV RNA synthesis emphasizing the role of the P, N and L VSV gene products during transcription.
FIG. 11 represents a schematic of an engineered VSV genome showing the sequence of the gene junctions (SEQ ID NO:8); any foreign gene flanked by gene end and start signals are faithfully transcribed by the viral polymerase.
FIG. 12 represents a schematic of the VSV-T7 RNA polymerase construct as described herein.
FIG. 13 shows expression of GFP in cells transfected with the vaccinia T7 system and pTM1-GFP plasmid encoding an IRES element.
FIG. 14 shows expression of GFP in cells transfected with the vaccinia T7 system and pSP73-GFP plasmid without an IRES element.
FIG. 15 shows expression of GFP in cells transfected with the VSV-T7 system and pTM1-GFP plasmid encoding an IRES element.
FIG. 16 shows expression of GFP in cells transfected with the VSV-T7 system and either the pSP73-GFP (no IRES) or pTM1-GFP (with IRES) plasmids.
FIG. 17 shows comparison of expression of GFP in cells transformed with either the VSV-T7 or vaccinia-T7 systems.
FIG. 18 shows expression of GFP in cells with the VSV-T7 system and co-transfected with plasmids pTM1-D1 and pTM1-D12 that encode the vaccinia capping enzyme.



DETAILED DESCRIPTION
A. Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter described herein belongs.
As used herein, the terms “heterologous” or “foreign nucleic acid” are used interchangeably and refer to DNA or RNA that does not occur naturally as part of the genome in which it is present or which is found in a location or locations in the genome that differs from that in which it occurs in nature. Heterologous nucleic acid is generally not endogenous to the cell into which it is introduced, but has been obtained from another cell or prepared synthetically. Generally, although not necessarily, such nucleic acid encodes RNA and proteins that are not normally produced by the cell in which it is expressed. Any DNA or RNA that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which it is expressed is herein encompassed by the term heterologous DNA. Examples of heterologous DNA include, but are not limited to, DNA that encodes transcriptional and translational regulatory sequences and selectable or traceable marker proteins, such as a protein that confers drug resistance. Heterologous DNA may also encode DNA that mediates or encodes mediators that alter expression of endogenous DNA by affecting transcription, translation, or other regulatable biochemical processes.
As used herein, the term “vector” refers to a polynucleotide construct designed for transduction/transfection of one or more cell types. Selection and use of such vectors are well within the level of skill of the art. Generally, vectors are derived from viruses or plasmids of bacteria and yeasts. As used herein, VSV vectors may be, for example, “cloning vectors” which are designed for isolation, propagation and replication of inserted nucleotides, “expression vectors” which are designed for expression of a nucleotide sequence in a host cell, a “viral vector” which is designed to result in the production of a recombinant virus or virus-like particle, or “shuttle vectors”, which comprise the attributes of more than one type of vector. The present invention encompasses VSV vectors that comprise nucleic acid encoding viral structural proteins capable of assembling into virus-like particles. As used herein, in the context of VSV, a “heterologous polynucleotide” or “heterologous gene” or “transgene” is any polynucleotide or gene that is not present in wild-type VSV. As used herein, in the context of VSV, a “heterologous” promoter is one which is not associated with or derived from VSV.
As used herein, “expression” refers to the process by which nucleic acid is transcribed into mRNA and translated into peptides, polypeptides, or proteins. “Expression” may be characterized as follows: a cell is capable of synthesizing many proteins. At any given time, many proteins which the cell is capable of synthesizing are not being synthesized. When a particular polypeptide, coded for by a given gene, is being synthesized by the cell, that gene is said to be expressed. In order to be expressed, the DNA sequence coding for that particular polypeptide must be properly located with respect to the control region of the gene. The function of the control region is to permit the expression of the gene under its control. As used herein, the term “expression vector” includes vectors capable of expressing DNA or RNA fragments that are in operative linkage with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA or RNA fragments. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA or RNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or may integrate into the host cell genome.
As used herein, the terms “operative linkage” or “operative association” of heterologous DNA to regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences, refer to the functional relationship between such DNA and such sequences of nucleotides. For example, operative linkage of heterologous DNA to a promoter refers to the physical and functional relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and correctly transcribes the DNA.
As used herein, the term “promoter region” refers to the portion of DNA of a gene that controls transcription of DNA to which it is operatively linked. A portion of the promoter region includes specific sequences of DNA that are sufficient for RNA polymerase recognition, binding and transcription initiation. This portion of the promoter region is referred to as the promoter. In addition, the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of the RNA polymerase. These sequences may be cis acting or may be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, may be constitutive or regulated. For use herein, inducible promoters are preferred. The promoters are recognized by an RNA polymerase that is expressed by the host. The promoter may be any bacteriophage promoter that can be recognized by an RNA polymerase that is expressed in the host.
As used herein, “RNA polymerase” may be endogenous to the host or may be introduced by genetic engineering into the host, either as part of the host chromosome or on an episomal element, including a plasmid containing the DNA encoding an RNA polymerase. The RNA polymerase may be a T7 polymerase derived from a prokaryotic source.
As used herein, the term “transcription terminator region” has (a) a subsegment that encodes a polyadenylation signal and polyadenylation site in the transcript, and/or (b) a subsegment that provides a transcription termination signal that terminates transcription by the polymerase that recognizes the selected promoter. The entire transcription terminator may be obtained from a protein-encoding gene, which may be the same or different from the gene, which is the source of the promoter. Transcription terminator regions can be those that are functional in E. coli. Transcription terminators are optional components of the expression systems herein, but are employed in preferred embodiments.
As used, the term “nucleotide sequence coding for expression of” a polypeptide refers to a sequence that, upon transcription and subsequent translation of the resultant mRNA, produces the polypeptide.
As used herein, the term “expression control sequences” refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus, expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, maintenance of the correct reading frame of a protein-encoding gene to permit proper translation of the mRNA, and stop codons. In addition, sequences of nucleotides encoding a fluorescent indicator polypeptide, such as a green or blue fluorescent protein, can be included in order to select positive clones (i.e., those host cells expressing the desired polypeptide).
As used herein, “host cells” or “target cells” are cells in which a vector can be propagated and its nucleic acid expressed. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Such progeny are included when the term “host cell” is used. A “host cell” includes an individual cell or cell culture which can be or has been a recipient of a VSV vector(s) of this invention. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected, (no permanent genetic change is possible in this system) or infected in vivo or in vitro with a VSV vector of this invention.
As used herein, the term “packaging cell” or “packaging cell line” refers to a cell or cell lines that are able to package viral genomes or modified genomes or its equivalents. Thus, packaging cells can provide complementing functions for any genes deleted in a viral genome (e.g. nucleic acids encoding structural genes) and are able to package the viral genomes into viral vector particles. The production of such particles requires that the genome be replicated and that those proteins necessary for assembling a viral particle (infectious or non-infectious) are produced. The particles also can require certain proteins necessary for the maturation of the viral particle. Such proteins can be provided by the vector or by the packaging cell.
As used herein, the term “transfection” refers to the taking up of DNA or RNA by a host cell. Transformation refers to this process performed in a manner such that the DNA is replicable, either as an extrachromosomal element or as part of the chromosomal DNA of the host. Methods and means for effecting transfection and transformation are well known to those of skill in this art (see, e.g., Wigler et al. (1979) Proc. Natl. Acad. Sci. USA 76:1373-1376; Cohen et al. (1972) Proc. Natl. Acad. Sci. USA 69:2110).
As used herein, the term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. A “host cell” is a cell that has been transformed, or is capable of transformation, by an exogenous nucleic acid molecule. Host cells containing the transformed nucleic acid fragments are referred to as “transgenic” cells, and organisms comprising transgenic cells are referred to as “transgenic organisms”.
As used herein, the term “substantially pure” means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis, or high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.
As used herein, the term “isolated substantially” pure DNA refers to DNA fragments purified according to standard techniques employed by those skilled in the art (see, e.g., Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
As used herein, a “culture” means a propagation of cells in a medium conducive to their growth, and all sub-cultures thereof. The term subculture refers to a culture of cells grown from cells of another culture (source culture), or any subculture of the source culture, regardless of the number of subculturings that have been performed between the subculture of interest and the source culture. The term “to culture” refers to the process by which such culture propagates.
As used herein, the term “peptide” and/or “polypeptide” means a polymer in which the monomers are amino acid residues which are joined together through amide bonds, alternatively referred to as a polypeptide. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. Additionally, unnatural amino acids such as beta-alanine, phenylglycine, and homoarginine are meant to be included. Standard single and three letter naming conventions for amino acids are used herein.
As used herein, the term “restriction enzyme digestion” of DNA refers to catalytic cleavage of the DNA with an enzyme that acts only at certain locations in the DNA. Such enzymes are called restriction endonucleases, and the sites for which each is specific is called a restriction site. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements as established by the enzyme suppliers are used. Restriction enzymes commonly are designated by abbreviations composed of a capital letter followed by other letters representing the microorganism from which each restriction enzyme originally was obtained and then a number designating the particular enzyme. In general, about 1 μg of plasmid or DNA fragment is used with about 1-2 units of enzyme in about 20 μl of buffer solution. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation of about 1 hour at 37° C. is ordinarily used, but may vary in accordance with the supplier's instructions. After incubation, protein or polypeptide is removed by extraction with phenol and chloroform, and the digested nucleic acid is recovered from the aqueous fraction by precipitation with ethanol. Digestion with a restriction enzyme may be followed with bacterial alkaline phosphatase hydrolysis of the terminal 5′ phosphates to prevent the two restriction cleaved ends of a DNA fragment from circularizing or forming a closed loop that would impede insertion of another DNA fragment at the restriction site. Unless otherwise stated, digestion of plasmids is not followed by 5′ terminal dephosphorylation. Procedures and reagents for dephosphorylation are conventional as described in Sections 1.56-1.61 of Sambrook, et. al., Molecular Cloning: A Laboratory Manual New York: Cold Spring Harbor Laboratory Press, 1989 (which disclosure is hereby incorporated by reference).
The terms “recovery” or “isolation” of a given fragment of DNA from a restriction digest mean separation of the digest, e.g., on polyacrylamide or agarose gel by electrophoresis, identification of the fragment of interest by comparison of its mobility versus that of marker DNA fragments of known molecular weight, removal of the gel section containing the desired fragment, and separation of the gel from DNA. These procedures are generally well known. For example, see Lawn et al., 1981, Nucleic Acids Res., vol. 9, pp. 6103-6114; and Goeddel et al., 1980, Nucleic Acids Res., vol. 8, p. 4057, which disclosures are hereby incorporated by reference.
As used herein, the term “gene” refers to those DNA sequences which transmit the information for and direct the synthesis of a single protein chain.
As used herein, “gene therapy” refers to genetic therapy that involves the transfer of heterologous DNA to certain cells, target cells, of a mammal, particularly a human, with a disorder or condition for which such a therapy is sought. The DNA is introduced into the selected target cells in a manner such that the heterolgous DNA is expressed and a therapeutic product encoded thereby is produced. Alternatively, the heterolgous DNA can in some manner mediate expression of DNA that encodes the therapeutic product, it can encode a product, such as a peptide or RNA that in some manner mediates, directly or indirectly, expression of a therapeutic product. Gene therapy also can be used to deliver nucleic acid encoding a gene product to replace a defective gene or supplement a gene product produced by the mammal or a cell in which it is introduced. The introduced nucleic acid can encode a therapeutic compound, such as a growth factor or inhibitor thereof, or a tumor necrosis factor or inhibitor thereof, or a receptor thereof, that is not normally produced in the mammalian host or that is not normally produced in therapeutically effective amounts or at a therapeutically useful time. The heterolgous DNA encoding the therapeutic product can be modified prior to introduction into the cells of the afflicted host to enhance or otherwise alter the product or expression thereof.
As used herein, “therapeutic nucleic acid” refers to a nucleic acid that encodes a therapeutic product. The product can be nucleic acid, such as a regulatory sequence or gene, or can be a protein that has a therapeutic activity or effect. For example, a therapeutic nucleic acid can be a ribozyme, antisense, double-stranded RNA, a nucleic acid encoding a protein or otherwise.
As used herein, the term “infection” refers to the invasion by agents (e.g., viruses, viral vector particles, bacteria, etc.) of cells where conditions are favorable for their replication and growth.
As used herein, the term “plasmid” means a vector used to facilitate the transfer of exogenous genetic information, such as the combination of a promoter and a heterologous gene under the regulatory control of that promoter. The plasmid can itself express a heterologous gene inserted therein. “Plasmids” are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are commercially available, are publicly available on an unrestricted basis, or can be constructed form such available plasmids in accord with published procedures. In addition, other equivalent plasmids are known in the art and will be apparent to one of ordinary skill in the art.
The term “ligation” means the process of forming phosphodiester bonds between two nucleic acid fragments. To ligate the DNA fragments together, the ends of the DNA fragments must be compatible with each other. In some cases, the ends will be directly compatible after endonuclease digestion to blunt ends to make them compatible for ligation. To blunt the ends, the DNA is treated in a suitable buffer for at least 15 minutes at 15° C., with about 10 units of the Klenow fragment of DNA polymerase I or T4 DNA polymerase in the presence of the four deoxyribonucleotide triphosphates. The DNA is then purified by phenolchloroform extraction and ethanol precipitation. The DNA fragments that are to be ligated together are put in solution in about equimolar amounts. The solution will also contain ATP, ligase buffer, and a ligase such as T4 DNA ligase at about 10 units per 0.5 g of DNA. If the DNA is to be ligated into a vector, the vector is first linearized by digestion with the appropriate restriction endonuclease(s). The linearized fragment is then treated with bacterial alkaline phosphatase, or calf intestinal phosphatase to prevent self-ligation during the ligation step.
As used herein, the term “preparation of DNA from cells” means isolating the plasmid DNA from a culture of the host cells. Commonly used methods for DNA preparation are the large and small scale plasmid preparations described in sections 1.25-1.33 of Sambrook et al., supra, which disclosure is hereby incorporated by reference. After preparation of the DNA, it can be purified by methods well known in the art such as that described in section 1.40 of Sambrook et al., supra, which disclosure is hereby incorporated by reference.
The terms “polynucleotide” and “nucleic acid”, used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include a single-, double- or triple-stranded DNA, genomic DNA, cDNA, genomic RNA, mRNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be a oligodeoxynucleoside phosphoramidate (P—NH2) or a mixed phosphoramidate-phosphodiester oligomer. Peyrottes et al. (1996) Nucleic Acids Res. 24: 1841-8; Chaturvedi et al. (1996) NucleicAcids Res. 24: 2318-23; Schultz et al. (1996) Nucleic Acids Res. 24: 2966-73. A phosphorothioate linkage can be used in place of a phosphodiester linkage. Braun et al. (1988) J. Immunol. 141: 2084-9; Latimer et al. (1995) Molec. Immunol. 32: 1057-1064. In addition, a double-stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer. Reference to a polynucleotide sequence (such as referring to a SEQ ID NOS:1-6) also includes the complement sequence.
The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, genomic RNA, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support.
As used herein, the term “under transcriptional control” refers to a term well understood in the art and indicates that transcription of a polynucleotide sequence depends on its being operably (operatively) linked to an element which contributes to the initiation of, or promotes, transcription. “Operably linked” refers to a juxtaposition wherein the elements are in an arrangement allowing them to function.
“Replication” and “propagation” are used interchangeably and refer to the ability of an VSV vector of the invention to reproduce or proliferate. These terms are well understood in the art. For purposes of this invention, replication involves production of VSV proteins and is generally directed to reproduction of VSV. Replication can be measured using assays standard in the art. “Replication” and “propagation” include any activity directly or indirectly involved in the process of virus manufacture, including, but not limited to, viral gene expression; production of viral proteins, nucleic acids or other components; packaging of viral components into complete viruses; and cell lysis.
As used herein, “expression” includes transcription and/or translation of a polynucleotide sequence.
As used herein, “internal ribosome entry sequence” (“IRES”) refers to nucleic acid sequences which exhibit IRES activity (IRES elements), i.e. sequences which are capable of providing cap-independent translation of a downstream gene or coding sequence by an internal ribosome entry mechanism.
As used herein, “functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA, siRNA, or other RNA that may not be translated but yet has an effect on at least one cellular process.
As used herein, “altered levels” refers to the level of expression in transgenic cells or organisms that differs from that of normal or untransformed cells or organisms.
As used herein, a first sequence is an “antisense sequence” with respect to a second sequence if a polynucleotide whose sequence is the first sequence specifically binds to, or specifically hybridizes with, the second polynucleotide sequence under physiological conditions.
As used herein, “antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of protein from an endogenous gene or a transgene.
As noted above, another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase “hybridizing specifically to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. “Bind(s) substantially” refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.
“Stringent conditions” or “high stringency conditions”, as defined herein, are identified by, but not limited to, those that (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C. “Moderately stringent conditions” are described by, but not limited to, those in Sambrook et al., supra, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
Polynucleotides are described as “complementary” to one another when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides. A double-stranded polynucleotide can be “complementary” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second. Complementarity (the degree that one polynucleotide is complementary with another) is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base-pairing rules.
As used herein, “RNA interference (RNAi)” is the process of sequence-specific, post-transcriptional gene silencing initiated by double stranded RNA (dsRNA) or siRNA. RNAi is seen in a number of organisms such as Drosophila, nematodes, fungi and plants, and is believed to be involved in anti-viral defense, modulation of transposon activity, and regulation of gene expression. During RNAi, dsRNA or siRNA induces degradation of target mRNA with consequent sequence-specific inhibition of gene expression.
As used herein, “interfering RNA” and “small interfering RNA” (siRNA) refer to a RNA duplex of nucleotides that is targeted to a gene of interest. A “RNA duplex” refers to the structure formed by the complementary pairing between two regions of a RNA molecule. siRNA is “targeted” to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to a nucleotide sequence of the targeted gene. In some embodiments, the length of the duplex of siRNAs is less than 30 nucleotides. In some embodiments, the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides in length. In some embodiments, the length of the duplex is 19-25 nucleotides in length. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure may contain a loop portion positioned between the two sequences that form the duplex. The loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The hairpin structure can also contain 3′ and/or 5′ overhang portions. In some embodiments, the overhang is a 3′ and/or a 5′ overhang 1, 2, 3, 4 or 5 nucleotides in length.
The siRNA can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter. The nucleic acid sequence can also include a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadelylation signal.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the scope of those of skill in the art. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Handbook of Experimental Immunology” (D. M. Weir & C. C. Blackwell, eds.); “Gene Transfer Vectors for Mammalian Cells” (J. M. Miller & M. P. Calos, eds., 1987); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987); “PCR: The Polymerase Chain Reaction”, (Mullis et al., eds., 1994); and “Current Protocols in Immunology” (J. E. Coligan et al., eds., 1991).
B. VSV-T7 RNA Polymerase Polypeptide Expression System
1. VSV
For general information related to vesicular stomatitis virus, see, “Fundamental Virology”, second edition, 1991, ed. B. N. Fields, Raven Press, New York, pages 489-503; and“Fields Virology”, third edition, 1995, ed. B. N. Fields, vol. 1, pages 1121-1159.
“VSV” as used herein refers to any strain of VSV or mutant forms of VSV, for example such as those described in WO 01/19380. A VSV construct of this invention may be in any of several forms, including, but not limited to, genomic RNA, mRNA, cDNA, part or all of the VSV RNA encapsulated in the nucleocapsid core, VSV complexed with compounds such as PEG and VSV conjugated to a nonviral protein. VSV vectors provided herein may encompass replication-competent and replication-defective VSV vectors, such as, VSV vectors lacking G glycoprotein of M glycoprotein. Replication-defective VSV vectors can be grown in appropriate cell lines. FIG. 7 represents a schematic of a vesicular stomatitis virus, including a representation of the VSV genome. General features of VSV RNA synthesis are illustrated in FIGS. 9 and 10.
a. VSV Sequences and Constructs
VSV, a member of the Rhabdoviridae family, is a negative-stranded virus that replicates in the cytoplasm of infected cells, does not undergo genetic recombination or reassortment, has no known transforming potential and does not integrate any part of it genome into the host. VSV comprises an about 11 kilobase genome that encodes for five proteins referred to as the nucleocapsid (N), polymerase proteins (L) and (P), surface glycoprotein (G) and a peripheral matrix protein (M). The genome is tightly encased in nucleocapsid (N) protein and also comprises the polymerase proteins (L) and (P). Following infection of the cell, the polymerase proteins initiate the transcription of five subgenomic viral mRNAs, from the negative-sense genome, that encode the viral proteins. The polymerase proteins are also responsible for the replication of the full-length viral genomes that are packaged into progeny virions. The matrix (M) protein binds to the RNA genome/nucleocapsid core (RNP) and also to the glycosylated (G) protein, which extends from the outer surface in an array of spike like projections and is responsible for binding to cell surface receptors and initiating the infectious process.
Following attachment of VSV through the (G) protein to receptor (s) on the host surface, the virus penetrates the host and uncoats to release the RNP particles. The polymerase proteins, which are carried in with the virus, bind to the 3′ end of the genome and sequentially synthesize the individual mRNAs encoding N, P, M, G, and L, followed by negative-sense progeny genomes. Newly synthesized N, P and L proteins associate in the cytoplasm and form RNP cores which bind to regions of the plasma membrane rich in both M and G proteins.
FIGS. 1 and 2 depict schematic illustrations of VSV-T7 RNA polymerase plasmids as provided herein and as set forth in SEQ ID NOS:1 and 2, respectively.
b. Viral Particles Form and Budding or Release of Progeny Virus Ensues.
A table of various VSV strains is shown in “Fundamental Virology”, second edition, supra, at page 490. WO01/19380 and U.S. Pat. No. 6,168,943 disclose that strains of VSV include Indiana, New Jersey, Piry, Colorado, Coccal, Chandipura and San Juan. The complete nucleotide and deduced protein sequence of a VSV genome is known and is available as Genbank VSVCG, accession number J02428; NCBI Seq ID335873; and is published in Rose and Schubert, 1987, in The Viruses: The Rhabdoviruses, Plenum Press, NY. pp. 129-166. A complete sequence of a VSV strain is shown in U.S. Pat. No. 6,168,943.
VSV New Jersey strain is available from the American Type Culture Collection (ATCC) and has ATCC accession number VR-159. VSV Indiana strain is available from the ATCC and has ATCC accession number VR-1421.
Provided herein are compositions and methods encompassing any form of VSV, including, but not limited to genomic RNA, mRNA, cDNA, and part or all of VSV RNA encapsulated in the nucleocapsid core. The present invention encompasses VSV in the form of a VSV vector construct as well as VSV in the form of viral particles. Also provided herein are nucleic acid encoding specific VSV vectors disclosed herein, such as set forth in SEQ ID NOS:1 and 2. As provided herein, VSV vectors encompass replication-competent as well as replication-defective VSV vectors. Replication-competent VSV viral particles were prepared using standard methodology such as described by Whelan et al. (Proc Natl Acad Sci USA. 1995 Aug. 29; 92(18):8388-92).
In certain aspects, the VSV vector lacks a protein function essential for assembly and release of infectious particles, such as G-protein function or M protein function. The VSV vector may lack several protein functions essential for replication. Such vectors are useful in producing VSV-T7 replication-defective viral particles. For example, plasmids encoding the M and G genes (pTM1-M and pTM1-G) can be co-transfected with standard plasmids encoding the L, P, and N proteins into vaccinia-T7 virus-infected BHK-21 cells. The co-transfected template plasmid in this case encodes the replication-defective virus genome, which includes the T7 RNA polymerase gene but not the M and G viral genes. Following initial packaging and release of the defective viral particles, further amplification of the defective particles is accomplished by co-transfection with pTM1-M and pTM1-G plasmids to supply the missing M and G proteins. In the absence of the complementing plasmids, replication-defective viruses replicate the defective genome and express N, P and L proteins in the infected cells, but the defective genomes are neither packaged nor released. Co-transfection of cells infected with VSV-T7 replication-defective viral vectors and with plasmids encoding a transgene leads to efficient protein expression of the transgene in the cell.
In certain embodiments, viral particles comprising a VSV vector provided herein encode the polynucleotide sequence for T7 RNA polymerase. Also provided is isolated nucleic acid encoding the recombinant VSV vector, as well as host cells comprising a recombinant VSV vector for producing such particles. Schematic illustrations of engineered VSV-T7 viral particle genomes are shown in FIGS. 11 and 12. Any foreign gene flanked by gene end and start signals are faithfully transcribed by the viral polymerase (see FIG. 11).
2. T7 RNA Polymerase Expression System
a. T7 RNA Polymerase and Promoter
As provided herein, the disclosed polypeptide expression system, involves infecting cultured cells with a VSV-T7 recombinant virus and transfecting these infected cells with a plasmid encoding a heterologous DNA sequence under control of the T7 promoter and an IRES element (see FIG. 12). The gene encoding T7 RNA polymerase is derived from a prokaryotic source. In one aspect, the VSV-T7 recombinant virus vector is engineered to express a prokaryotic T7 RNA polymerase enzyme in the cytoplasm of infected cells. The infected cells are then transfected with a recombinant plasmid vector encoding a heterologous DNA downstream of a T7 promoter sequence and an IRES element. This results in cytoplasmic accumulation of large amounts of T7 mRNA transcripts which are efficiently translated into the desired protein.
In another aspect, the T7 RNA polymerase enzyme in the cytoplasm of infected cells is capable of transcription of a heterologous DNA downstream of a T7 promoter sequence, but without an IRES element. In such circumstances, the cell is co-transfected with plasmids expressing the two subunits of the vaccinia virus-capping enzyme under control of the virus, as discussed in detail below.
As provided herein, a T7 promoter sequence facilitates binding of T7 polymerase to a polynucleotide sequence for the initiation of transcription. Also contemplated are any bacteriophage promoter sequences that can be recognized by an RNA polymerase that is expressed in the host.
b. IRES
T7 transcripts synthesized using the VSV-T7 expression system provided herein lack cap structures at their 5′ ends and thus require an IRES element for efficient translation. The IRES element is capable of providing cap-independent translation of a downstream gene or coding sequence by an internal ribosome entry mechanism. In certain aspects, recombinant plasmid vectors provided herein encode a T7 promoter sequence, an IRES element and a heterologous polynucleotide sequence for efficient transcription and expression of the desired protein.
c. Vaccinia Virus-Capping Enzymes
To circumvent the requirement for an IRES element in the plasmid encoding the heterologous polynucleotide of interest provided herein are compositions and methods for co-transfecting target cells with plasmid vectors encoding the D1 and D12 subunits of vaccinia virus-capping enzyme, as set forth in SEQ ID NOS:5 and 6. Expression of the vaccinia virus-capping enzyme results in capping of the 5′ end of nascent transcripts, thereby facilitating efficient translation. The capping enzyme sequences may be provided as separate plasmid vectors.
3. Recombinant Plasmids
Recombinant plasmids provided herein were constructed using standard molecular biology cloning techniques known to those of ordinary skill in the art. Recombinant VSV viruses that express T7 RNA polymerase can drive expression of the desired heterologous polynucleotide sequence encoded on plasmids under control of a T7 promoter. Other bacteriophage promoter sequences may also be used. FIG. 3 illustrates plasmid pTM1-GFP (SEQ ID NO:3) encoding an IRES element and the green fluorescent protein (GFP) reporter gene. The GFP gene from pBI-GFP was inserted into the NcoI site of the pTM-1 vector using standard restriction enzyme cloning techniques. As will be recognized by one of skill in the art, the GFP gene can be replaced by any heterologous polynucleotide sequence using appropriate restriction enzyme recognition sites and standard molecular biology techniques.
FIG. 4 illustrates plasmid pSP73-GFP (SEQ ID NO:4) encoding the GFP reporter gene, but without an IRES element. The GFP gene from pBI-EGFP vector was inserted into the EcoRI site of the pSP73 vector. As noted above, the GFP can be replaced by any heterologous nucleotide sequence using standard restriction enzyme cloning techniques.
4. Expression of Polypeptides
As described herein, the VSV-T7 expression system is used to infect a cell with VSV-T7 viral vector particles followed by transfection of recombinant plasmids encoding a transgene using techniques well known to those of skill in the art (see, e.g., Wigler et al. (1979) Proc. Natl. Acad. Sci. USA 76:1373-1376; Cohen et al. (1972) Proc. Natl. Acad. Sci. USA 69:2110). One embodiment as described herein, involves infecting cultured cells with a VSV-T7 recombinant virus and transfecting these infected cells with a plasmid encoding a heterologous polynucleotide sequence (e.g. transgene) under control of the T7 promoter and an IRES element. Although VSV normally shuts off host cell protein synthesis, it was found that T7 transcripts are efficiently translated under these conditions to yield protein amounts comparable to the vaccinia-T7 system. T7 transcripts synthesized in this VSV-T7 system lack cap structures at their 5′ end and thus require an IRES element for translation. In another embodiment provided herein, compositions and methods are provided wherein VSV-T7 infected cells are co-transfected with plasmids encoding the two subunits of the vaccinia virus-capping enzyme (SEQ ID NOS:5 and 6) under control of an IRES element, thereby circumventing the requirement for an IRES element in the plasmid encoding the transgene of interest. Also contemplated is a defective VSV-T7 recombinant virus that lacks virus-encoded host cell shutoff functions for improving expression of the transgene.
4. Expression of Interfering RNAs
Provided herein is a viral vector expression system for delivery of a nucleic acid sequence encoding a small interfering RNA molecule (siRNA) targeted against a gene of interest.
The target gene may be a gene derived from the cell, an endogenous gene, a transgene, or a gene of a pathogen which is present in the cell after infection thereof. Depending on the particular target gene and the dose of double stranded RNA material delivered, the procedure may provide partial or complete loss of function for the target gene. A reduction or loss of gene expression in at least 99% of targeted cells has been shown. Lower doses of injected material and longer times after administration of dsRNA may result in inhibition in a smaller fraction of cells. Quantitation of gene expression in a cell may show similar amounts of inhibition at the level of accumulation of target mRNA or translation of target protein.
The RNA may comprise one or more strands of polymerized ribonucleotide. The double-stranded structure may be formed by a single self-complementary RNA strand, by two complementary RNA strands or by co-transfecting cells with plasmids encoding transgenes or fragments thereof in opposite orientation relative to the T7 promoter. RNA duplex formation may be initiated either inside or outside the cell. The RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses of double-stranded material may yield more effective inhibition. Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition. RNA containing a nucleotide sequence identical to a portion of the target gene can be used for inhibition. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Thus, sequence identity may be optimized by alignment algorithms known in the art and calculating the percent difference between the nucleotide sequences. Alternatively, the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript.
The cell with the target gene may be derived from or contained in any organism (e.g., plant, animal, protozoan, virus, bacterium, or fungus). RNA may be synthesized either in vivo or in vitro. Cloned RNA polymerase as provided by the VSV-T7 expression system may mediate transcription in vivo. For transcription from a transgene in vivo or an expression construct, a regulatory region, i.e., the T7 promoter, may be used to transcribe the RNA strand (or strands) (the regulatory sequence is the T7 promoter). A method of reducing the expression of a gene product in a cell, comprising contacting a cell with the VSV viral particle encoding T7 RNA polymerase and appropriate recombinant plasmid vector encoding the desired interfering RNA nucleotide sequence are also provided
RNA interference is now established as an important biological strategy for gene silencing, but its application to mammalian cells has been limited by nonspecific inhibitory effects of long double-stranded RNA on translation. Provided herein are compositions and methods for a viral mediated delivery mechanism that results in delivery of small interfering RNA (siRNA) into a target cell. This viral mediated strategy is generally useful in reducing expression of target genes in order to model biological processes or to provide therapy for dominant human diseases.
EXAMPLES
Example 1
Vaccinia-T7 Expression System
High level expression of a reporter protein (green fluorescent protein) using an IRES-containing plasmid construct was accomplished using the VSV-T7 expression system provided herein and shown to be comparable in efficiency to the vaccinia-T7 system. Infectious VSV-T7 viral particles were recovered from BHK-321 cells (baby hamster kidneys) transfected with pVSV-T7 (SEQ ID NO:1; FIG. 1) grown in minimal essential medium (MEM) supplemented with 7% newborn calf serum using standard tissue culture techniques. Supernatants containing viral particles were titered by plaque assay and used as inoculae for expression studies. Vaccinia-T7 virus stocks were prepared using similar techniques in BSC-40 cells (monkey kidney). All expression experiments were performed using nearly confluent BHK-21 cells grown in monolayers in 5 cm plates ( ̃1×106 cells per plate). All virus adsorptions were performed at a multiplicity of 10 pfu/cell in a volume of 0.3 ml for 1 h at room temperature. Vaccinia-T7 inoculae also included 10 μg/ml of DEAE-dextran and 40 μg/ml Ara-C.
Following adsorption, the virus inoculae were removed before addition of 0.5 ml of the transfection reagents containing 45 μl lipofection reagent (prepared as described by Rose et al. (Biotechniques 1991, 10:520-525)) and 455 μA of MEM containing various amounts of plasmid encoding the reporter gene. After 1 hr, 1.5 ml of fresh MEM plus 7% newborn calf serum was added (containing 40 μg/ml Ara-C for vaccinia-T7 infections) and monolayers were incubated at 37° C. Expression of GFP was examined by fluorescent microscopy at various times.
FIG. 13 shows expression of GFP in cells infected with vaccinia-T7 viral particles, followed by transfection with 11 μg of pTM1-GFP (with IRES), at 4 h, 9 h, and 12 h, post-transfection (bottom panel). Cells infected with vaccinia-T7 viral particles and then transfected with 11 μg of pSP73-GFP (no IRES) are shown in FIG. 14 (EGF expressing cells shown in the bottom panel). The level of GFP expression is similar with or without an IRES element encoded in the expression plasmid when using a vaccinia-T7 viral expression system.
Example 2
VSV-T7 Expression System
FIG. 15 shows GFP expression in cells infected with VSV-T7 viral particles and transfected with 11 μg of pTM1-GFP (with IRES) at 6 h, 9 h and 12 h post-transfection (bottom panel). Expression of the same reporter gene without an IRES element, as shown in FIG. 16, indicates that the VSV-T7 system requires an IRES element for efficient expression of the transgene if 5′ capping enzymes are not provided.
Example 3
Comparison of Vaccinia-T7 and VSV-T7 Expression Systems
A comparison of the VSV-T7 expression system with the vaccinia-T7 system indicates similar levels of reporter gene expression when cells are transfected with plasmids encoding an IRES (FIG. 17, bottom panel). As a comparison, cells infected with VSV viral particles encoding GFP instead of T7 RNA polymerase also express GFP at comparable levels (far right panels).
Example 4
VSV-T7 Expression System with Vaccinia Virus-Capping Enzymes
To circumvent the requirement of an IRES element in the plasmid driving expression of the transgene, cells were co-transfected with plasmids encoding the D1 and D12 subunits of the vaccinia virus-capping enzyme under control of an IRES element (SEQ ID NOS:5 and 6; FIGS. 5 and 6). Capping functions at the 5′ end of the transcript are therefore provided in trans, thus bypassing the need for an IRES element. GFP expression of cells infected with VSV-T7 viral particles followed by co-transfection with pSP73-GFP (no IRES), pTM1D1 and pTM1-D12 plasmids is shown in FIG. 18. These results indicate that the VSV-T7 expression system can be used successfully without the requirement of an IRES element if the transgene 5′ capping functions are provided in trans.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

## Claims

1. A recombinant vesicular stomatitis virus vector expression system comprising
(a) a VSV comprising a polynucleotide encoding a T7 RNA polymerase or other bacteriophage RNA polymerase and the D1 subunit of vaccinia capping enzyme, wherein when the vector is used to infect a cell, the RNA polymerase and the D1 subunit of vaccinia capping enzyme are expressed in the cell;
(b) a recombinant plasmid vector comprising a polynucleotide comprising the following elements operably linked 5′ to 3′:
(i) a bacteriophage promoter corresponding to the bacteriophage RNA polymerase, and
(ii) a heterologous gene, wherein when the recombinant plasmid vector is used to transfect said cell, a polypeptide is expressed from transcripts encoded by said heterologous gene in the cell.

2. The system of claim 1, wherein the VSV comprises a polynucleotide encoding a T7 RNA polymerase and wherein the bacteriophage promoter is a T7 promoter.

3. The system of claim 1, wherein the recombinant plasmid vector comprises a T7 promoter corresponding to residues 794 to 813 of SEQ ID NO:3, and a heterologous gene, with IRES polynucleotide encoding sequences deleted.

4. The system of claim 1, wherein the VSV is encoded by a vector that comprises (a) the polynucleotide sequence set forth in SEQ ID NO:1, and (b) a polynucleotide encoding the sequence for the D1 catalytic subunit of vaccinia virus-capping enzyme as set forth in SEQ ID NO:5.

5. The system of claim 4, with the proviso that the M and/or G genes of the VSV are deleted or mutated such that the VSV is replication-deficient.

6. The system of claim 1, wherein the VSV further comprises a polynucleotide encoding the D12 subunit of vaccinia capping enzyme, wherein when the vector is used to infect a cell, the D12 subunit of vaccinia capping enzyme is expressed in the cell.

7. The system of claim 6, wherein the VSV comprises a polynucleotide encoding the sequences for D1 and D12 catalytic subunits of vaccinia virus-capping enzyme as set forth in SEQ ID NOS:5 and 6.

8. The system of claim 6, with the proviso that the M and/or G genes of the VSV are deleted or mutated such that the VSV is replication-deficient.

9. The system of claim 1, wherein said cells were transfected with said recombinant plasmid vectors by liposome mediated transfer, lipofection, polycation-mediated transfer, or direct DNA transfer or uptake.